CRYO X-RAY MICROSCOPY WITH HIGH-SPATIAL-RESOLUTION IN AMPLITUDE AND PHASE-CONTRAST

The resolution of transmission X-ray microscopes (TXMs) using zone pla te optics is presently about 30 nm. Theory and experiments presented h ere show that this resolution can be obtained in radiation sensitive h ydrated biological material by using shock frozen samples. For this pu rpose the interaction of X-rays with matter and the image formation wi th zone plates is described. For the first time the influence of the l imited apertures of the condenser and the zone plate objective are in included in calculations of the image contrast, the photon density and radiation dose required for the object illumination. Model considerat ions show that lowest radiation dose and high image contrast are obtai ned in optimized phase contrast which exploits absorption as well as p hase shift. The damaging effect of the absorbed X-rays is quantitative ly evaluated by radiation-induced kinetics showing that cryogenic samp les are structurally stable. To verify these theoretical models the TX M was modified to allow imaging of frozen-hydrated samples at atmosphe ric pressure. Details inside cells and algae as small as 35 nm are vis ible at 2.4 nm wavelength in amplitude contrast mode. At this resoluti on the cryogenic samples show no structural changes. As predicted, opt imized phase contrast shows structures inside the frozen-hydrated obje cts with high contrast. Stereo-pair images of algae reveal the 3D orga nization of the organelles. Element analysis and micro-tomography of w hole cryogenic cells are possible. (C) 1998 Elsevier Science B.V. All rights reserved.